Targeted Neuronal Ablation Using Two-Photon Microscopy in a Zebrafish Larava

Begin with an anesthetized transgenic zebrafish larva immobilized in agarose and placed in a Petri dish containing an anesthetic solution.

Position it under a two-photon laser microscope, which uses two photons for detailed imaging.

Focus on the top and bottom layers of the brain to set imaging boundaries.

Capture images at various depths to observe neurons before laser treatment.

Adjust the focus on the region of interest and adjust the frame size to refine the focus on neurons.

Select and irradiate the neurons using the two-photon laser.

The Laser induces localized heat, resulting in neuronal death with minimal damage to surrounding tissue. Capture the image.

Compare pre and post-treatment images to assess fluorescence changes.

Refocus neurons in deeper planes and repeat the irradiation to treat all neurons in the region of interest..

Recapture the image and compare it with the pre-treatment image. The loss of fluorescence after laser treatment confirms successful neuronal ablation.

Place the agarose-embedded larva under a 2-photon laser-scanning microscope and start the microscopy system. Open the image acquisition software, and under the Laser tab, click the on button to turn on the las